Specific Growth Factors Research Articles

Autologous induced pluripotent stem cell-derived four-organ-chip.

Microphysiological systems play a pivotal role in progressing toward a global paradigm shift in drug development. Here, we designed a four-organ-chip interconnecting miniaturized human intestine, liver, brain and kidney equivalents. All four organ models were predifferentiated from induced pluripotent stem cells from the same healthy donor and integrated into the microphysiological system. The coculture of the four autologous tissue models in one common medium deprived of tissue specific growth factors was successful over 14-days. Although there were no added growth factors present in the coculture medium, the intestine, liver and neuronal model maintained defined marker expression. Only the renal model was overgrown by coexisting cells and did not further differentiate. This model platform will pave the way for autologous coculture cross-talk assays, disease induction and subsequent drug testing.

A review on transcriptional regulation responses to hypoxia in mesenchymal stem cells.

Mesenchymal stem cells (MSCs), which are known for having therapeutic applications, reside in stem cell niches where the oxygen concentration is low. At the molecular level, the master regulator of the cellular reaction to hypoxia is hypoxia-inducible transcription factor (HIF). The transcriptional response of a cell to hypoxia is affected by two major components; first, the structure of hypoxia-response elements (HREs), which primarily define how much of the HIF signal is integrated into the transcriptional output of individual genes. Second, the availability of other transcriptional factors cooperating with HIF in the context of HRE. In MSCs, the expression of a single gene by hypoxia depends on elements such as factors influencing the HIF activity, metabolic pathways, the real oxygen concentration in the cellular microenvironment, and duration of culture. In addition, specific growth factors and pro-infection cytokines, hormones, oncogenic signaling, as well as ultrasound are potent regulators of HIF in MSCs. Altogether, the response of MSCs to hypoxia is complex and mediated by several genes and molecular agents. Regarding the influence of hypoxia on MSCs, oxygen concentration must be taken into consideration based on the cell type and the aim of culture before a particular MSCs culture.

Bioactive glass functionalized chondroitin sulfate hydrogel with proangiogenic properties.

Blood vessels play an important role in bone defect repair and growth, and a critical challenge of bone defect repair is the promotion of blood vessel formation. Most of the current methods promote vascularization by adding specific growth factors, which are costly and easy to inactivate. In this study, we developed a covalently cross-linked aminated bioactive glass nanoparticle-chondroitin sulfate methacrylate (ABGN-CSMA) organic-inorganic composite hydrogel with angiogenic properties. The amino groups of the ABGNs form covalent bonds with the carboxyl groups on CSMA. Surface amination modification of BGNs not only improved the dispersion of BGNs in CSMA but also significantly improved the mechanical properties of the composite hydrogel. The largest storage modulus (1200 Pa), the largest loss modulus (560 Pa) and the strongest resistance to deformation of the hydrogel are seen at 10% concentration of ABGNs. Simultaneously, the local pH stability and sustained ion release of the composite hydrogel are conducive to cell adhesion, proliferation, and angiogenesis. This work provides evidence for the development of covalently cross-linked organic-inorganic composite hydrogels with angiogenic properties.

3D printed HUVECs/MSCs cocultures impact cellular interactions and angiogenesis depending on cell-cell distance

Vascularization is a crucial process during the growth and development of bone 1, yet it remains one of the main challenges in the reconstruction of large bone defects. The use of in vitro coculture of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) has been one of the most explored options. Both cell types secrete specific growth factors that are mutually beneficial, and studies suggested that cell-cell communication and paracrine secretion could be affected by a number of factors. However, little is known about the effect of cell patterning and the distance between cell populations on their crosstalk. In the present study, we showed that the separation and distance between ECs and MSCs populations affects angiogenesis by modulating cell-cell communication. HUVECs grown farther apart from MSCs (˃400 μm) presented characteristics of an early stage of angiogenesis (migration/proliferation). Results showed an increase in the up-regulation of VEGF, FGF-2, and ITGA3 (integrins) but a smaller fold change in the expression of VE-Cadherin and Ang-1. HUVECs were also still highly proliferative. On the contrary, HUVECs incubated closer (≤200 μm) to MSCs, showed signs of stabilization, mainly an increase in Ang-1 and VE-cadherin expression, as well as tighter monolayers. Conditioned media collected from HUVECs and MSCs grown ≤200 μm apart preferentially promoted tube formation, a later stage of angiogenesis, due in part to a significant increase in Ang-1 paracrine secretion. In addition, in groups in which fibers were printed farther apart (400 μm), cells produced EVs with a significantly increase cargo. Finally, in vivo experiment results showed an increase in blood vessels density and new bone thickness after 12 weeks of implantation in rat cranial defect, further suggesting the higher efficiency of indirect ECs/MSCs contact in prompting the release of paracrine signals that stimulate the angiogenesis of local tissues, and enhanced subsequent bone regeneration.

Fully Synthetic Heparan Sulfate-Based Neural Tissue Construct That Maintains the Undifferentiated State of Neural Stem Cells

Heparin and heparan sulfate (HS) are attractive components for constructing biomaterials due to their ability to recruit and regulate the activity of growth factors. The structural and functional heterogeneity of naturally derived heparin and HS is, however, an impediment for the preparation of biomaterials for regenerative medicine. To address this problem, we have prepared hydrogels modified by well-defined synthetic HS-derived disaccharides. Human induced pluripotent cell-derived neural stem cells (HIP-NSCs) encapsulated in a polyethylene glycol-based hydrogel modified by a pendent HS disaccharide that is a known ligand for fibroblast growth factor-2 (FGF-2) exhibited a significant increase in proliferation and self-renewal. This observation is important because evidence is emerging that undifferentiated stems cells can yield significant therapeutic benefits via their paracrine signaling mechanisms. Our data indicate that the HS disaccharide protects FGF-2, which has a very short biological half-live, from degradation. It is anticipated that, by careful selection of a synthetic HS oligosaccharide, it will be possible to control retention and release of specific growth factor, which in turn will provide control over cell fate.

Osteoblasts grown on microroughened titanium surfaces regulate angiogenic growth factor production through specific integrin receptors

Cellular attachment and response to biomaterials are mediated by integrin receptor binding to extracellular matrix proteins adsorbed onto the material surface. Osteoblasts interact with their substrates via several integrin complexes including fibronectin-binding α5β1 and collagen-binding α1β1 and α2β1. Knockdown of α2 or β1 integrin subunits inhibits the production of factors that promote an osteogenic microenvironment, including osteocalcin, osteoprotegerin, and TGFβ1. Osteoblasts also secrete several angiogenic growth factors such as VEGF-A (VEGF165), FGF-2, and angiopoietin 1, which are regulated by titanium surface topography and surface energy. Here, we examined whether signaling through integrin receptor complexes regulates production and secretion of angiogenic factors during osteoblast differentiation on microtextured Ti surfaces. To do this, integrin subunits α1, α2, α5, and β1 were stably silenced in MG63 osteoblast-like cells cultured on grit-blasted/acid-etched hydrophobic Ti (SLA) or on hydrophilic SLA (modSLA). VEGF-A production increased in response to Ti surface topography and energy in integrin α2, α5, and β1 silenced cells but decreased in α1-silenced cells. FGF-2 decreased on modSLA substrates in both α1 and α2-silenced cells but was unchanged in response to silencing of either α5 or β1. In integrin α1, α2, and β1-silenced cells, Ang-1 increased on modSLA but α5-silencing did not affect Ang-1 production during surface mediated differentiation. These results suggest that signaling through specific integrin receptor complexes during osteoblast differentiation on microstructured Ti substrates, regulates the production of angiogenic factors by those cells, and this is differentially regulated by surface hydrophilicity. Statement of SignificanceSuccessful implantation of synthetic biomaterials into bone depends on the biological process known as osseointegration. Osseointegration is a highly regulated communication of cells that orchestrates the migration of progenitor cells towards the implant site and promotes the deposition and mineralization of extracellular matrix proteins within the implant microenvironment, to tightly join the implant to native bone. In this process, angiogenesis functions as the initiation site of progenitor cell migration and is necessary for matrix deposition by providing the necessary nutrients for bone formation. In the present study, we show a novel regulation of specific angiogenic growth factors by integrin receptor complexes. This research is important to develop biomaterials that promote and maintain osseointegration through proper vascularization and prevent implant failure in patients lacking sufficient angiogenesis.

Abstract 1039: c-Met activation promotes epithelial-to-mesenchymal transition (EMT) in renal cancer cells through the upregulation of the chemokine receptor CXCR7

Abstract Purpose: The purpose of this study is to explore how the receptor tyrosine kinase (RTK) c-Met modulates the expression of the pro-tumorigenic molecule CXCR7, a chemokine receptor, in renal cancer cells; and to investigate the role of c-Met and CXCR7 in promoting the epithelial-mesenchymal transition (EMT) of renal cancer cells. Background: c-Met, a pro-oncogenic RTK, can play a major role in the survival and progression of cancer cells; and it is often overexpressed in both clear cell and papillary renal cell carcinoma (RCC). c-Met, upon activation by its specific ligand hepatocyte growth factor (HGF), can induce a broad spectrum of biological pathways involved in the growth of cancer cells. CXCR family of chemokine receptors and their chemokine ligands were shown to exhibit decisive roles in tumor initiation, promotion, progression, and metastasis. CXCR7 is upregulated in several types of cancers, including renal cancer, and found to be involved in tumor cell development, survival, and metastasis. A global process integral to the cancer metastasis is EMT, which results in the disruption of cell-to-cell junctions and endows epithelial cells with the ability to migrate and invade surrounding tissue. EMT process is associated with an increase of mesenchymal markers like vimentin and a decrease of epithelial markers like E-cadherin in cancer cells. Sustained activation of c-Met induces EMT in renal cancer cells, however, the cross-talk between CXCR7 and c-Met signaling in the promotion of renal cancer EMT remains unclear. Results: We assessed the expression levels of CXCR7 by Western blot analysis and found that CXCR7 is overexpressed in human renal cancer cells (both 786-O and ACHN) when compared to normal renal epithelial cells (RPTEC). Also, in both 786-O and ACHN cells, HGF-induced activation of c-Met further increased the expression of CXCR7 at both RNA and protein levels. In renal cancer cells (both 786-O and ACHN), siRNA-mediated knockdown of c-Met or treatment with XL-184 (a small molecule inhibitor of c-Met) inhibited c-Met-induced upregulation of CXCR7. c-Met activation increased the expression of pro-EMT markers such as vimentin, N-cadherin, and MMP-9 and suppressed the expression of anti-EMT molecule E-cadherin, as measured by Western blot and flow cytometry. On the other hand, siRNA-mediated silencing of CXCR7 attenuated the induction of EMT, suggesting that CXCR7 plays a major role in c-Met-induced EMT in renal cancer cells. Conclusion: Together, our findings demonstrate that the c-Met-CXCR7 axis can trigger the EMT, which can play an important role(s) in renal cancer proliferation and migration. Citation Format: Samik Chakraborty, Murugabaskar Balan, Soumitro Pal. c-Met activation promotes epithelial-to-mesenchymal transition (EMT) in renal cancer cells through the upregulation of the chemokine receptor CXCR7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1039.

Abstract 3116: Targeting fibroblast growth factor receptors in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma and is classified into two main histopathological subtypes: embryonal RMS (eRMS), characterized by different genomic changes or alveolar RMS (aRMS), driven by the oncogenic fusion protein PAX3-FOXO1. The significant toxicity associated with conventional chemotherapies represents a major complication in pediatric oncology. To improve current therapies, we adopted two different strategies targeting fibroblast growth factor receptors (FGFR) in RMS. FGFR1-4 are a family of transmembrane receptor tyrosine kinases. Their activation upon binding of fibroblast growth factors (FGF) triggers pro-survival and proliferative signals. Our goal is to deliver drugs specifically to the tumor site by taking advantage of FGFR4 overexpression in RMS. To this end, we will covalently link FGFR4 specific nanobodies to the surface of liposomal vincristine in order to actively target RMS cells. We have established the optimal conditions to formulate liposomes loaded with vincristine. Following nanobody phage display selection on recombinant FGFR4 we focused on the top scoring candidates. Flow cytometry analysis on FGFR4-expressing versus FGFR4 knock-out RMS cell lines showed receptor-specific binding of three nanobodies. In activation assays with the FGFR4 specific growth factor FGF19, we demonstrated that the three binding candidates also blocked downstream ERK activation in RMS cells. We will now assess their theranostic potential on drug-loaded and fluorescently labeled nanovesicles on RMS tumor cells in vitro and in xenografts in vivo. Surprisingly, we observed change in cell morphology followed by cell death upon exposure to FGF2 in a subset of cultured cells established from eRMS patient-derived xenografts. Inappropriate expression of FGFRs and FGF signaling is implicated in tumor progression and therefore our findings appear contradictory. Dose-response experiments have shown that FGFR inhibition with small molecule inhibitors completely rescued FGF2 toxicity. In contrast, however, we detected high expression levels of FGFR1, 2 and 4 as well as activating mutations of FGFR4 in FGF2-sensitive eRMS cells. Therefore, our results are of upmost clinical relevance since genetically-based drug selection could lead to an inappropriate treatment inducing tumor promoting conditions. Hence, our second goal is to further unravel the molecular mechanism underlying the toxic effect of FGF-2 in a subset of eRMS tumors to avoid potentially harmful treatments. In summary, we have identified FGFR4 specific nanobodies that bind to the receptor and block downstream signaling in RMS cells. Active drug delivery of liposomal vincristine to the tumor site has the potential to enhance the therapeutic impact and decrease side effects. Moreover, we discovered a toxic effect of FGF2 in a subgroup of eRMS patient derived xenograft cells which might open new avenues for treatment. Citation Format: Nagjie Alijaj, Sandrine Moutel, Maxim Gray, Maurizio Roveri, Gabriele Manzella, Marco Wachtel, Franck Perez, Beat Schäfer, Michele Bernasconi. Targeting fibroblast growth factor receptors in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3116.

Pulmonary Arterial Hypertension Due to NPR-C Mutation: A Novel Paradigm for Normal and Pathologic Remodeling?

Idiopathic Pulmonary Arterial Hypertension (IPAH) is a deadly and disabling disease characterized by severe vascular remodeling of small pulmonary vessels by fibroblasts, myofibroblasts and vascular smooth muscle cell proliferation. Recent studies suggest that the Natriuretic Peptide Clearance Receptor (NPR-C) signaling pathways may play a crucial role in the development of IPAH. Reduced expression or function of NPR-C signaling in pulmonary artery smooth muscle cells may contribute to the pulmonary vascular remodeling, which is characteristic of this disease. The likely mechanisms may involve an impaired interaction between NPR-C, specific growth factors and other signal transduction pathways including but not limited to Gqα/mitogen-activated protein kinase (MAPK)/PI3K and AKT signaling. The resulting failure of growth suppression in pulmonary artery smooth muscle cells provides critical clues to the cellular pathobiology of IPAH. The reciprocal regulation of NPR-C signaling in models of tissue remodeling may thus provide new insights to our understanding of IPAH.

VEGF – Supplemented extracellular matrix is sufficient to induce endothelial differentiation of human iPSC

Extracellular matrix (ECM) provides a scaffold for cells and tissues, but also supports organogenesis and tissue remodeling. The required instructive properties of the ECM to interact with cells depend on matrix architecture, structural proteins and functional matrix components such as growth factors, providing spatial, chemical and functional cues. Decellularized ECM (dECM) has been proposed as an instructive material that promotes tissue regeneration. We investigated the instructive ECM elements preserved in dECM and necessary to promote endothelial differentiation of human induced pluripotent stem cells (hiPSC). We show that detergent-decellularized human kidney ECM remains structurally intact and carries a number of heparin-binding growth factors, including FGF2, VEGF, BMP2, HGF, EGF, PDGF-BB and TGFβ, albeit at reduced levels compared to native tissues. Clearance of these heparin-binding factors, or heparan-sulfate proteoclycans from ECM resulted in massively reduced differentiation of hiPSC, suggesting that remaining structural dECM proteins such as laminin, collagen or fibronectin alone are not instructive. In contrast, replenishing dECM with VEGF replaced medium-supplemented VEGF and resulted in more efficient differentiation of hiPSC into endothelial cells, and even in the absence of other culture-supplemented differentiation factors dECM alone was superior to geltrex. In conclusion, conditioning of dECM with specific growth factors acting as functional cues may allow to generate functional niches by selective promotion of cell attachment, survival and differentiation.

Levels of glial cell line-derived neurotrophic factor are decreased, but fibroblast growth factor 2 and cerebral dopamine neurotrophic factor are increased in the hippocampus in Parkinson's disease.

Growth factors can facilitate hippocampus-based learning and memory and are potential targets for treatment of cognitive dysfunction via their neuroprotective and neurorestorative effects. Dementia is common in Parkinson's disease (PD), but treatment options are limited. We aimed to determine if levels of growth factors are altered in the hippocampus of patients with PD, and if such alterations are associated with PD pathology. Enzyme-linked immunosorbent assays were used to quantify seven growth factors in fresh frozen hippocampus from 10 PD and nine age-matched control brains. Western blotting and immunohistochemistry were used to explore cellular and inflammatory changes that may be associated with growth factor alterations. In the PD hippocampus, protein levels of glial cell line-derived neurotrophic factor were significantly decreased, despite no evidence of neuronal loss. In contrast, protein levels of fibroblast growth factor 2 and cerebral dopamine neurotrophic factor were significantly increased in PD compared to controls. Levels of the growth factors epidermal growth factor, heparin-binding epidermal growth factor, brain-derived neurotrophic factor and mesencephalic astrocyte-derived neurotrophic factor did not differ between groups. Our data demonstrate changes in specific growth factors in the hippocampus of the PD brain, which potentially represent targets for modification to help attenuate cognitive decline in PD. These data also suggest that multiple growth factors and direction of change needs to be considered when approaching growth factors as a potential treatment for cognitive decline.

PS1236 COST-EFFECTIVENESS AND COST-UTILITY ANALYSIS OF MULTIPLE TREATMENT STRATEGIES USING ABVD AND/OR BEACOPP IN THE TREATMENT OF ADVANCED-STAGE HODGKIN LYMPHOMA

Background: Donor-derived red blood cells (RBCs) are the most common form of cellular therapy, coupled with donor-dependency, alloimmunization and blood borne disease risks. In vitro derivation of RBCs from an immortal source, as iPSCs provide an alternative solution to these issues. In addition, in vitro iPSC-RBCs allow genome editing for possible gene therapies, while large scale production would be beneficial in RBC drug delivery. The current limitation of iPSC-RBCs is their development immaturity, leading to low enucleation potential, which requires improvement prior clinical application. Aims: We aim to push iPSC-derived erythroid differentiation to definitive erythropoiesis yielding enucleated erythrocytes Methods: A minimum of three independent iPSC lines were used during the studies. These iPSC lines were single cell seeded and emerging colonies were differentiated towards erythroblasts using specific growth factor cocktails and media developed at Sanquin. Human mononuclear cells isolated from fetal liver, cord blood and adult blood were also differentiated to erythroblasts. Flow cytometry was used to assess culture purity, stage of erythropoiesis and enculeation. RT-PCR and HPLC was used to quantify the developmentally defined globins. RNA was isolated from the different erythroblasts and RNA-sequencing was performed and analysed. Single cell RNA-seq was performed on single cell FACS-sorted iPSC derived hematopoietic cells. Results: To further develop the process, we have established a GMP-compatible iPSC generation and differentiation protocol, that upon hematopoietic specification yielded erythroblasts (EBL). This method resulted in 2–6∗105 EBLs/iPSC with 100% CD71+/CD235+ (erythroid-specific markers). Further maturation yielded orthochromatic normoblasts containing a mix of primitive and definitive erythroid waves, leading to poor enucleation potential and/or reticulocyte stability. Total RNAseq analysis comparing iPSC to in vitro derived definitive erythroid cells from fetal liver, cord and adult revealed significant differences and identified specific genes that may be used to further augment iPSC-erythroid terminal differentiation to erythrocytes. For instance, iPSC-derived erythroblasts lack expression of specific erythroid regulators (e.g. MYB and KLF1 target genes SOX6, BCL11A and BCAM). Single-cell RNAseq of early iPSC differentiation was performed, aiming to recognize distinct waves of hematopoiesis. Five cell clusters were identified of which, one was HSPC-like, 3 lineage-committed populations and 1 of unknown origin. This data coupled with index sorting, defined the HSPCs as CD71+/CD235−, and the erythroid cells as CD71+/CD235+ expressing embryonic and fetal Hbs. In addition, we have found differentially expressed markers between the iPSC-RBCs and definitive erythroid cells that may be used to better define specific erythroid waves during iPSC differentiations and/or fetal development. We hypothesize, that the identified HSPCs potentially give rise to developmentally more mature cells, which we aim to expand. Currently we are pursuing different approaches among which forward programming, to support early iPSC-HSPCs that produce developmentally more mature definitive erythroid cells including proper enucleation potential. Summary/Conclusion: In conclusion, we have performed extensive RNA analysis of iPSC, cord, fetal and adult erythroblast and identified potential targets that may aid in proper iPSC-derived erythroid differentiation to enucleated red cells.

M2 receptors activation modulates cell growth, migration and differentiation of rat Schwann-like adipose-derived stem cells

Schwann cells (SCs) play a central role in peripheral nervous system physiology and in the response to axon injury. The ability of SCs to proliferate, secrete growth factors, modulate immune response, migrate and re-myelinate regenerating axons has been largely documented. However, there are several restrictions hindering their clinical application, such as the difficulty in collection and a slow in vitro expansion. Adipose-derived stem cells (ASCs) present good properties for peripheral nerve regenerative medicine. When exposed to specific growth factors in vitro, they can acquire a SC-like phenotype (dASCs) expressing key SCs markers and assuming spindle-shaped morphology. Nevertheless, the differentiated phenotype is unstable and several strategies, including pharmacological stimulation, are being studied to improve differentiation outcomes. Cholinergic receptors are potential pharmacological targets expressed in glial cells. Our previous work demonstrated that muscarinic cholinergic receptors, in particular M2 subtype, are present in SCs and are able to modulate several physiological processes. In the present work, muscarinic receptors expression was characterised and the effects mediated by M2 muscarinic receptor were evaluated in rat dASCs. M2 receptor activation, by the preferred agonist arecaidine propargyl ester (APE), caused a reversible arrest of dASCs cell growth, supported by the downregulation of proteins involved in the maintenance of cell proliferation and upregulation of proteins involved in the differentiation (i.e., c-Jun and Egr-2), without affecting cell survival. Moreover, M2 receptor activation in dASCs enhances a pronounced spindle-shaped morphology, supported by Egr2 upregulation, and inhibits cell migration. Our data clearly demonstrate that rat dASCs express functional muscarinic receptors, in particular M2 subtype, which is able to modulate their physiological and morphological processes, as well as SCs differentiation. These novel findings could open new opportunities for the development of combined cell and pharmacological therapies for peripheral nerve regeneration, harnessing the potential of dASCs and M2 receptors.

Effects of Nel-like molecule-1 and bone morphogenetic protein 2 combination on rat pulp repair.

Nel-like molecule-1 (NELL-1) is a novel highly specific growth factor that can induce osteoblast differentiation and bone formation as well as odontoblast differentiation. Recent studies have suggested that NELL-1 can synergistically increase bone formation and regeneration with bone morphogenetic protein 2 (BMP2) and inhibit adverse effects induced by BMP2. This study aimed to evaluate the combined effects of NELL-1 and BMP2 on rat pulp repair. The experiment used healthy non-carious maxillary first molars from 60 Wistar rats. Exposed pulps were capped with NELL-1 plus BMP2, NELL-1 alone, and BMP2 alone, and each was absorbed onto a sterile collagen sponge. In the control samples, the collagen sponge alone and Dycal were used as capping agents. After l, 2 and 4weeks, the rats were sacrificed. The formation of reparative dentin, as well the situation of pulp repair, was detected by hematoxylin-eosin (HE) staining; moreover, the expression of dentin specific protein-dentin sialophosphoprotein (DSPP) and the pro-inflammatory cytokines interleukin-6 (IL6) and interleukin-8 (IL8) was detected by immunohistochemical staining. Quantitative real-time PCR experiment was used to investigate the mRNA levels of IL6 and IL8. The results showed that pulp capping with NELL-1 plus BMP2 in rats had superior ability in inducing reparative dentin formation with dentin tubules and in reducing the inflammatory cell response compared with the other groups. These findings suggested that combined use of NELL-1 and BMP2 could positively regulate pulp repair.

Peripheral blood derived stem cells in OA knee at low resource setting: a phase II RCT

Purpose: Osteoarthritis (OA) of the knee is one of the main causes of musculoskeletal disability. It is clinically heterogeneous, and the processes that cause deterioration are still poorly understood. Arthritis is now often considered in terms of organ failure. Because of limitations in the effectiveness of conventional management options, alternative options such as biological and regenerative methods are coming into vogue. Current research efforts are focused on the identification of key biochemical pathways that can be targeted therapeutically through biological intervention and the testing of protein bio-therapeutics for restoring the metabolic balance within the joint. In particular, the most recent knowledge regarding tissue biology highlights the potential use of specific growth factors as therapeutic proteins for cartilage repair, and this is now being widely investigated in vitro and in vivo. Nevertheless, the complex OA process involves interplay of several growth factors needed in joint homeostasis and cartilage metabolism. OA of knee is one of the most common of all musculoskeletal complaints for which reason patients seek physician consultation at PM&R department of BSMMU. This prospective clinical trial is designed to evaluate the role of peripheral blood derived stem cell therapy in the late stages of knee OA. Methods: This phase II pilot RCT is being conducted after the IRB ethical clearance memo No. bsmmu/2018/25. All the patients attending PM&R department having primary OA Knee (Kellgren-Lawrence grade III and IV) are being considered as the sample. Among them who fulfill the selection criteria are being enrolled as study subjects after obtaining informed written consent. Joint ultrasonography is be done to measure cartilage thickness for all respondents. All respondents are being divided into two groups by using randomization technique. In Control group, total 15 respondents will receive standard care for Knee Osteoarthritis and in Case group, total 15 patients will be treated with single dose autologous peripheral blood derived stem cells therapy and conventional conservatives. Stem cells are being harvested using Granulocytes Colony Stimulating Factor (G-CSF) 30MU for consecutive 05 days and CD34, CD145 stem cells are being collected through apheresis. Quality is being ensured measuring cell surface antigen. The study is expected to be completed by June 2019. Results: During this reporting period, 6 patients were enrolled in each group. The patients is being assessed at the weeks of 0, 4, 12 and 24 for pain improvement in terms of VAS and function improvement in terms of (Western Ontario and McMaster Universities Arthritis Index [WOMAC] scoring and . In addition, these patients are being followed by post-treatment ultrasonography at 3 and 6 months. Among all respondents (n=12), Female 67% Mean Age: 53±11, Grade III: 84%. After 24 weeks of stem cell therapy, pain and functional significant improvement was observed and the mean cartilage thickness increased by 0.17 mm. Conclusions: Peripheral blood derived autologous stem cells are effective to improve quality of life. More large scale research is warranted to confer it.

Expression and purification of AtRALF1 from Escherichia coli

Rapid Alakalinization Factors (RALFs) are a family of plant specific peptide growth factors involved in myriad plant processes including growth, development, and response to stresses. These secreted peptides are genetically encoded and processed into an active form from a larger pre-pro-peptide. The active forms are ~5 kDa and have two disulfide bridges. Here we report our attempts to express the active form of AtRALF1 (At1g02900) from the soluble fractions of two different expression strains of E. coli, Origami 2 (DE3) and SHuffleT7 pLysY. Both of these strains are commercially available and genetically altered for expression of proteins with disulfide bonds. Expression of these peptides is challenging because misfolded disulfides or other structural issues result in insoluble peptides. We sought to optimize expression conditions to bulk purify HIS(6x)-AtRALF1 by cobalt column chromatography. Under most conditions the peptide was insoluble. However, at 25oC around 50% of total HIS(6x)-AtRALF1 was soluble in Origami cells. As controls, we expressed mutated versions with alterations in the conserved YISY motif which are physiologically inactive. Surprisingly, these mutant versions were 100% soluble in most conditions tested. All versions of HIS(6x)-AtRALF1 were observed to migrate ~12 kDa in SDS-PAGE gels although their predicted molecular weights is ~8 kDa. Treatment under strong reducing conditions had no effect on this migration pattern. We conclude that AtRALF1’s insolubility issues could be more related to a structure in the YISY motif than to the formation of disulfide bonds.

Identification of novel fibroblast-like cells from stem cells from human exfoliated deciduous teeth.

This study aimed to differentiate and characterize fibroblast-like cells from stem cells from human exfoliated deciduous teeth (SHED). The differentiation of fibroblast-like cells from SHED was carried out by using specific human recombinant connective tissue growth factor (CTGF). To characterize fibroblastic differentiation, the induced cells were subjected to morphological changes, proliferation rate, gene expression analysis using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), flow cytometry, and immunofluorescence staining. The commercial primary human gingival fibroblasts served as positive control in this study. The results from characterization analysis were compared with that of commercial cells to ensure that the cells differentiated from SHED were fibroblast-like cells. The results showed the inductive effect of CTGF for fibroblastic differentiation in SHED. SHED-derived fibroblasts were successfully characterized despite having similar morphological appearance, i.e., (i) significant proliferation rate between fibroblast-like cells and SHED, (ii) high expression of fibroblast-associated markers in qRT-PCR analysis, and (iii) positive staining against collagen type 1, fibroblast-specific protein 1, and human thymic fibroblasts in flow cytometry analysis and immunofluorescence staining. The same expression patterns were found in primary human gingival fibroblasts, respectively. SHED as negative control showed lower expression or no signal, thus confirming the cells differentiated from SHED were fibroblast-like cells. Taken together, the protocol adopted in this study suggests CTGF to be an appropriate inducer in the differentiation of SHED into fibroblast-like cells. The fibroblast-like cells differentiated from SHED could be used in future in vitro and in vivo dental tissue regeneration studies as well as in clinical applications where these cells are needed.

Application Value of Combined Detection of Urinary Amino Acid Metabolite and Tumor Specific Growth Factor in Evaluation of Curative Efficacy for Acute Leukemia

To investigate the clinical importance of combined detection of urinary amino acid metabolite --urinary tyrosine (UT) and tumor specific growth factor (TSGF) in the disease observation and curative effect evaluation of acute leukemia（AL）. In 87 cases of AL, the UT and TSGF levels were detected by using chemical chromogenic method and continuous monitoring method respectively. The 2 indicators of each treatment group were statistically analyzed and compared with that of 50 healthy control group. In 87 AL patients, the levels of UT and TSGF after treatment were significantly lower than those before treatment (P<0.01). The level of UT in non-remission(NR) group and partial remission(PR) group was significantly higher than that in completely remission(CR) group(P<0.01，P<0.05) and control group (P<0.01, P<0.01), The UT level in NR group was significantly higher than that in PR group (P<0.05), the UT level in CR group was significantly higher than that in control group (P<0.05). The levels of TSGF in NR and PR groups were significantly higher than those in CR and control groups(P<0.01, P<0.01). The level of TSGF in NR group was significantly higher than that in PR group(P<0.05), but no significant difference existed between CR group and control group(P>0.05). UT and TSGF levels are related with the metabolism of tumor cells in AL patients. UT level can be used to predict the recurrence of patients with CR. The combined dynamical monitoring of these 2 indicators can be used as the indexes for observation of AL status, evaluation of therapeutic efficacy, prediction of prognosis and relapse of AL.

Innovative Biomaterials for Bone Regrowth.

The regenerative medicine, a new discipline that merges biological sciences and the fundamental of engineering to develop biological substitutes, has greatly benefited from recent advances in the material engineering and the role of stem cells in tissue regeneration. Regenerative medicine strategies, involving the combination of biomaterials/scaffolds, cells, and bioactive agents, have been of great interest especially for the repair of damaged bone and bone regrowth. In the last few years, the life expectancy of our population has progressively increased. Aging has highlighted the need for intervention on human bone with biocompatible materials that show high performance for the regeneration of the bone, efficiently and in a short time. In this review, the different aspects of tissue engineering applied to bone engineering were taken into consideration. The first part of this review introduces the bone cellular biology/molecular genetics. Data on biomaterials, stem cells, and specific growth factors for the bone regrowth are reported in this review.

Endothelial Cell Growth Promoting Activity in Graves’ Disease Sera is Neutralized by Anti-Basic Fibroblast Growth Factor Antibodies in Patients with Fat Expansive but Not Infiltrative Orbitopathy

To report a case of orbital fat expansion leading to globe prolapse in a Graves' disease patient undergoing high-dose glucocorticoid therapy. To evaluate the growth factor receptor specificities of plasma autoantibodies in Graves' disease patients who exhibited contrasting subtypes of thyroid-associated ophthalmopathy, i.e. orbital fat expansion-type vs. infiltrative. Sera from Graves' orbitopathy and control patients with or without Graves' disease were subjected to protein-A affinity chromatography to obtain immunoglobulin G. A (1/50th to 1/1600th) range in dilutions of the protein-A eluate fraction was incubated for four days at 37 degrees C with bovine pulmonary artery endothelial cells to test for endothelial cell inhibition or stimulation. Growth stimulatory autoantibodies were co-incubated with specific neutralizing anti-insulin like growth factor 1 receptor antibodies or anti-basic fibroblast growth factor antibodies to assess autoantibody specificity in contrasting Graves' orbitopathy subtypes. We observed increased mean endothelial cell growth promoting activity in the protein-A eluates of serum from eighteen patients with active Graves' disease (117 ± 28%, n = 18) compared to mean endothelial cell activity (89 ± 10%, n = 13, P = 0.003) in thirteen adults without Graves' disease. The protein-A eluate fraction in acute infiltrative-type Graves' orbitopathy contained a high titer (> 1:1000) of endothelial cell stimulatory activity which was significantly neutralized by specific monoclonal anti-human insulin-like growth factor 1 receptor antibodies. The protein-A eluate fraction in fat expansion-type Graves' orbitopathy contained endothelial cell inhibitory activity (at low titers) and stimulatory activity (at high titers), and the latter stimulatory activity was completely neutralized by specific anti-basic fibroblast growth factor antibodies. Graves' disease suffering globe prolapse secondary to marked orbital fat-expansion had coexisting plasma fibroblast growth factor-inhibitory and -stimulatory autoantibodies. The latter was completely neutralized by anti-basic fibroblast growth factor antibodies.